Aeromonas salmonicida and infectious haematopoietic necrosis bivalent subunit vaccine and application thereof

Abstract

The invention discloses an aeromonas salmonicida and infectious hematopoietic necrosis bivalent subunit vaccine and application thereof, and belongs to the technical field of aquatic vaccines. The vaccine is characterized in that it comprises a fusion protein and an immunologic adjuvant. The fusion protein comprises a VapA protein antigen of aeromonas salmonicida and a G protein antigen of infectious haematopoietic necrosis virus, the VapA protein antigen and the G protein antigen are connected through a flexible Linker, and the preferable structure of the fusion protein is VapA-Linker-IHNV-G-6 * His. The invention also discloses a preparation method of the vaccine. The preparation method comprises the following steps: constructing a fusion protein expression vector, transforming a host, inducing expression, purifying the fusion protein and emulsifying with an adjuvant. The bivalent vaccine disclosed by the invention can simultaneously stimulate dual specific immune response aiming at bacteria and viruses, has a remarkable immune protection effect on aeromonas salmonicida and infectious hematopoietic necrosis viruses, is high in safety as a subunit vaccine, and provides an effective new way for prevention and control of multi-pathogen cross infection in aquaculture.

Description

Technical Field

[0001] This application relates to the field of aquatic vaccines, specifically to a bivalent subunit vaccine of Aeromonas salmonicida and infectious hematopoietic necrosis virus and its application. Background Technology

[0002] Aeromonas salmonidosis is a bacterial infectious disease caused by Aeromonas salmonidus, primarily resulting in scabies in fish. It is distributed globally, particularly in cold freshwater areas and coastal marine environments. The pathogenicity of Aeromonas salmonidus relies mainly on its various virulence factors, including outer membrane proteins, exotoxins, cytotoxins, and secretion systems. Currently, antibiotics are the primary treatment, but long-term antibiotic use is leading to increasing drug resistance and poses risks such as drug residues and environmental pollution. Vaccines are an effective means of prevention, and various vaccine studies, including inactivated and live attenuated vaccines, have been reported; however, the development of safer and more effective vaccines remains.

[0003] Infectious hematopoietic necrosis disease (IHH) is a viral fish disease caused by the infectious hematopoietic necrosis virus (IHV). It primarily affects salmonids and has a high mortality rate in juvenile fish. The disease is listed as a reportable animal disease by the World Organisation for Animal Health (OIE). Currently, research is underway on DNA vaccines, inactivated vaccines, and recombinant protein vaccines for this disease; however, due to factors such as cost, production scale, and technological barriers, vaccine rollout still faces challenges.

[0004] Currently, there are no vaccines in China that can simultaneously prevent both Aeromonas salmonellus and infectious hematopoietic necrosis virus. Traditional aquatic vaccines target only a few pathogens, and repeated immunizations can cause severe stress reactions. Therefore, developing bivalent or multivalent vaccines is one of the effective ways to address aquaculture diseases caused by cross-infection of multiple pathogens. Summary of the Invention

[0005] To address the current technical problem of the lack of vaccines in China that can simultaneously prevent two pathogens, Aeromonas salmonicidae and infectious hematopoietic necrosis virus, this invention discloses a bivalent subunit vaccine for Aeromonas salmonicidae and infectious hematopoietic necrosis virus and its application.

[0006] This invention discloses a bivalent subunit vaccine against Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV), comprising a fusion protein of Aeromonas salmonicida antigen and IHNV antigen, and an immune adjuvant. This vaccine, by fusing the antigens of the two pathogens into a single antigen, can simultaneously prevent infection by both pathogens. The design of the fusion protein allows the vaccine to stimulate an immune response against both pathogens, enhancing the protective effect. The immune adjuvant enhances the immune response and improves the immunogenicity of the vaccine.

[0007] Preferably, the structure of the above fusion protein is VapA-Linker-IHNV-G-6×His. The specific sequence is shown in SeqAAVapA-IHNV: 。

[0008] In this structure, VapA is the outer membrane protein of Aeromonas salmonidae, which has good immunogenicity; IHNV-G is a glycoprotein of infectious hematopoietic necrosis virus, which contains multiple antigenic epitopes and is stable; the Linker is a flexible linker peptide that can maintain the appropriate spatial conformation of the two proteins, which is conducive to the full exposure of the antigenic epitopes; the 6×His tag facilitates protein purification and detection.

[0009] Preferably, the Aeromonas salmonidae antigen and the infectious hematopoietic necrosis virus antigen are linked by a flexible linker. The flexible linker can reduce the steric hindrance between the two proteins, allowing them to maintain a relatively independent spatial conformation, which is beneficial for the exposure and recognition of their respective antigenic epitopes.

[0010] Preferably, the flexible linker described above is GGGGS. This sequence is a commonly used flexible linker peptide with good flexibility and hydrophilicity, which can effectively link two proteins without affecting their structure and function.

[0011] Preferably, the vaccine comprises 50-150 μg / mL of fusion protein and adjuvant ISA763. Within this concentration range, the fusion protein can adequately stimulate the immune response, while the adjuvant ISA763 can effectively enhance the immune effect and improve the protective efficacy of the vaccine.

[0012] This invention also provides a method for preparing a bivalent subunit vaccine against Aeromonas salmonicida and infectious hematopoietic necrosis, comprising the following steps: constructing a fusion protein expression vector, transforming an expression host, inducing expression of the fusion protein, purifying the fusion protein, and emulsifying the purified fusion protein with an adjuvant. This method constructs a fusion protein expression vector using molecular biology techniques, utilizes an expression host for protein expression, and obtains the vaccine through purification and emulsification. This method is simple to operate, suitable for large-scale production, and can obtain high-purity fusion proteins, ensuring the quality and efficacy of the vaccine.

[0013] Preferably, the induction conditions are: temperature 28℃, IPTG concentration 0.4mM, and induction time 18h. Under these conditions, the expression level of the fusion protein is highest, and the proportion of soluble expression is relatively high, which is beneficial for subsequent purification and application.

[0014] Preferably, the above-mentioned purified fusion protein includes the use of Ni 2+ Purification was performed using an affinity chromatography column. The His tag in the fusion protein was used to bind with Ni... 2+ Its affinity allows for efficient purification of target proteins, resulting in high-purity fusion proteins.

[0015] Preferably, the construction of the fusion protein expression vector involves inserting the VapA gene and the IHNV-G gene into the expression vector PGEX-4T-1 using homologous recombination technology. Homologous recombination technology is a highly efficient gene cloning method that can accurately insert the target gene into the expression vector, ensuring the correct expression of the fusion protein.

[0016] Compared with the prior art, the present invention has the following significant advantages: 1. Achieved Dual Immunoprotection: The recombinant fusion protein of this invention successfully combines protective antigens of bacteria (Aeromonas salmonicidae) and viruses (IHNV). Animal immunization experiments have demonstrated that the vaccine prepared from this protein can effectively stimulate rainbow trout to produce a strong immune response, including significantly upregulating the expression of multiple immune-related genes, producing high titers of anti-IHNV neutralizing antibodies, and providing up to 100% relative immune protection against Aeromonas salmonicidae challenge. This indicates that this invention provides, for the first time, a truly effective bivalent subunit vaccine capable of simultaneously protecting against two important pathogens.

[0017] 2. Solved key process challenges and improved manufacturability: This invention, through innovative extension and sequence optimization of the peptide linker connecting VapA and G proteins, successfully solved the serious technical defects of extremely low affinity purification efficiency caused by the coating of purification tags due to spatial conformational errors in the fusion protein. The optimized fusion protein purification yield was significantly improved, removing a key obstacle to the stable and large-scale production of the vaccine and giving it the potential for commercial development.

[0018] 3. High safety and broad application prospects: As a subunit vaccine, this invention does not contain live pathogens, making it far safer than attenuated live vaccines or inactivated whole bacterial / viral vaccines, avoiding the risk of virulence reversion. Its application will effectively reduce the use of antibiotics in aquaculture, reduce drug resistance problems, and is of great significance for ensuring food safety and environmental protection. Attached Figure Description

[0019] Figure 1 This is the predicted structure result of the VapA protein in this invention.

[0020] Figure 2 For the positive transformation verification of this invention, M: DL 5000 Maker; lanes 1-5: positive transformation is verified using specific primers p-for / p-rev; lanes 6-1: positive transformation is verified using specific primers PGEX-for / PGEX-rev.

[0021] Figure 3The images show the induced expression results of the recombinant vectors pVapA-IHNVuv and PGEX-4T-1-VapA-IHNV of this invention; where: M: 5-245 kDa protein molecular weight standard; 1: BL21 empty vector induction supernatant; 2: BL21 empty vector induction precipitation; 3: recombinant vector pVapA-IHNVuv induction supernatant; 4: recombinant vector pVapA-IHNVuv induction precipitation; 5: recombinant vector PGEX-4T-1-VapA-IHNV induction supernatant; 6: recombinant vector PGEX-4T-1-VapA-IHNV induction precipitation.

[0022] Figure 4 The Western Blot identification results of the recombinant vectors pVapA-IHNVuv and PGEX-4T-1-VapA-IHNV of this invention are shown below; where: M: 5-245 kDa protein molecular weight standard; 1: BL21 empty vector induction supernatant; 2: BL21 empty vector induction precipitation; 3: recombinant vector pVapA-IHNVuv induction supernatant; 4: recombinant vector pVapA-IHNVuv induction precipitation; 5: recombinant vector PGEX-4T-1-VapA-IHNV induction supernatant; 6: recombinant vector PGEX-4T-1-VapA-IHNV induction precipitation.

[0023] Figure 5 The results of the recombinant vector of the present invention induced expression at different temperatures are shown below; wherein: M: 15-130 kDa protein molecular weight standard; 1: 16℃ induction supernatant; 2: 16℃ induction precipitation; 3: 20℃ induction supernatant; 4: 20℃ induction precipitation; 5: 28℃ induction supernatant; 6: 28℃ induction precipitation; 7: 32℃ induction supernatant; 8: 32℃ induction precipitation; 9: 36℃ induction supernatant; 10: 36℃ induction precipitation.

[0024] Figure 6 The results of the recombinant vector of this invention induced expression at different inducer concentrations are shown below; where: M: 15-130 kDa protein molecular weight standard; 1: 0 mM induction supernatant; 2: 0.2 mM induction supernatant; 3: 0.4 mM induction supernatant; 4: 0.6 mM induction supernatant; 5: 0.8 mM induction supernatant; 6: 1.0 mM induction supernatant; 7: 0 mM induction precipitation; 8: 0.2 mM induction precipitation; 9: 0.4 mM induction precipitation; 10: 0.6 mM induction precipitation; 11: 0.8 mM induction precipitation; 12: 1.0 mM induction precipitation.

[0025] Figure 7The results of the recombinant vector of the present invention induced expression at different induction times are shown below; where: M: 15-130 kDa protein molecular weight standard; 1: supernatant after 0 h of induction; 2: precipitate after 0 h of induction; 3: supernatant after 6 h of induction; 4: precipitate after 6 h of induction; 5: supernatant after 12 h of induction; 6: precipitate after 12 h of induction; 7: supernatant after 18 h of induction; 8: precipitate after 18 h of induction; 9: supernatant after 24 h of induction; 10: precipitate after 24 h of induction.

[0026] Figure 8 This is the standard curve of absorbance versus protein concentration for this invention, where the x-axis represents protein concentration (mg / ml), the y-axis represents absorbance OD562, and the standard curve y = 1.0309x - 0.1101, R0 2 =0.9985.

[0027] Figure 9 The results of the kidney immune gene assay in this invention are as follows: IL-1β immune gene assay results. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0028] Figure 10 The results of the kidney immune gene assay in this invention are as follows: TLR-3 immune gene assay results. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0029] Figure 11 The results of the kidney immune gene assay of this invention are shown. The immune gene vig assay results are as follows: * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0030] Figure 12 The results of the kidney immune gene assay in this invention are shown in the stat assay results. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0031] Figure 13 The results of the kidney immune gene assay in this invention are shown below. The immune gene mx assay results are shown below. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0032] Figure 14 The results of the kidney immune gene assay of this invention are shown below. The results of the immune gene igM assay are shown below. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01).

[0033] Figure 15 The results of serum neutralizing antibody assay 28 days after immunization according to this invention are shown. * indicates a significant difference from the control group (P<0.05); ** indicates an extremely significant difference from the control group (P<0.01). Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0035] Example 1: Construction and Expression of Fusion Protein (1) Gene acquisition and fusion design: The VapA protein gene of Aeromonas salmonicida and the IHNV-G protein gene of Infectious Hematopoietic Necrosis Virus were obtained from the NCBI website.

[0036] like Figure 1 As shown, the three-dimensional structure and signal peptide of the VapA protein were predicted using AlphaFold and SignalP software, confirming the presence of a signal peptide at the N-terminus of the VapA protein.

[0037] like Figure 2 As shown, a recombinant protein was constructed by fusing with the C-terminus of the VapA protein. The structure is VapA-Linker-IHNV-G-6×His, where Linker is the flexible linker peptide GGGGS and 6×His is the purification tag.

[0038] (2) Primer design and PCR amplification: Specific primers V-for / V-rev and G-for / G-rev were designed and used as templates for PCR amplification with Aeromonas salmonidae C4 and YBEL-IHNV-G vector, respectively.

[0039] Design homologous recombination primers pV-for / V-Linker-rev, Linker-G-for / G-His-rev / pG-rev and PGEX-V-for / V-Linker-rev, Linker-G-for / G-His-rev / G-PGEX-rev to modify the target fragment. The PCR reaction system consisted of: 12.5 μL Primer Star Mix, 1 μL DNA, 1 μL forward primer, 1 μL reverse primer, 9.5 μL ddH2O, and a total volume of 25 μL.

[0040] The reaction program was as follows: 98℃ pre-denaturation for 60 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 16 s, 35 cycles, and a final extension at 72℃ for 5 min.

[0041] The fragment size of the product was determined by 1% gel electrophoresis, and the product was recovered using the GEL / PCR Purification Mini Kit.

[0042] (3) Construction of expression carrier: Glycerol-containing bacteria of pGFPuv and PGEX-4T-1 vectors were streaked onto LB agar plates containing AMP (100 μg / mL) and incubated at 37°C until single colonies formed.

[0043] Select a single colony and add it to 5 mL of LB liquid medium. Incubate overnight at 37°C with shaking. Extract plasmids using a plasmid miniprep kit.

[0044] The vector was linearized using EcoRI and XbaRI restriction enzymes. The reaction mixture consisted of 2 μL of 10×Fast Drgest Buffer, 4 μL of DNA, 1 μL of EcoRI, 1 μL of XbaRI, and 13 μL of ddH2O, for a total volume of 20 μL. The reaction conditions were 37℃ for 30 min and 80℃ for 5 min.

[0045] The gene fragments of VapA and IHNV-G were ligated to the linearized vector using a seamless cloning kit. The reaction system consisted of 5 μL of One Step Fusion Cloning Mix (2×), 2 μL of the insert, 1 μL of the linearized vector, and 2 μL of ddH2O, for a total volume of 10 μL. The reaction conditions were 50℃ for 15 min.

[0046] The reaction product was transformed into Eoli-DH5α competent cells, heat-shocked at 42°C for 30 s, incubated on ice for 2 min, 500 μL of LB medium was added, and the cells were incubated at 37°C with shaking for 1 h. The cells were then spread onto AMP-containing resistant LB plates and cultured overnight at 37°C.

[0047] Single colonies were selected for positive transformation screening using primers p-for / p-rev and PGEX-for / PGEX-rev. Positive templates with specific bands were sent to Qingdao Paisennuo Biotechnology Co., Ltd. for whole plasmid sequencing, and the sequence was SeqVapA-IHNV.

[0048] like Figure 3 As shown, the positive recombinants with correct sequencing results were inoculated into 5 mL of LB liquid medium, cultured overnight, and then the plasmid was extracted, the concentration was detected, and stored at -20℃ for later use.

[0049] (4) Identification of recombinant protein expression: like Figure 4 As shown, the recombinant vectors pVapA-IHNVuv and PGEX-4T-1-VapA-IHNV were transformed into Escherichia coli BL21, and positive transformants were screened using primers.

[0050] Positive transformants and BL21 empty vectors were inoculated into 300 mL LB liquid medium and cultured at 37°C and 180 rpm until the absorbance was about 0.5. Then, IPTG inducer with a final concentration of 0.1 mM was added and induced at 28°C and 180 rpm for 12 hours.

[0051] Collect 20 mL of bacterial culture by centrifugation, add 20 mL of PBS buffer, and sonicate for 2 hours using an ultrasonic disruptor (70% power, disrupt for 2 seconds, pause for 2 seconds).

[0052] Centrifuge to separate the supernatant and precipitate. Add 20 mL of PBS to the precipitate and vortex. Take 60 μL of the supernatant and 60 μL of the resuspended precipitate and mix with 20 μL of loading buffer. Heat at 100℃ for 10 min and centrifuge at 12000 rpm for 5 min. Collect the supernatant for SDS-PAGE electrophoresis and Western Blot analysis.

[0053] SDS-PAGE: Add 20 μL of protein sample to a 10% precast gel, electrophoresis at 160 V for 50 min, stain with Coomassie brilliant blue protein staining solution for 2 h, and destain with pure water until completely stained.

[0054] Western Blot: Cut the protein gel to the appropriate size and place it in transfer buffer; activate the PVDF membrane with methanol for 30 seconds, rinse with deionized water for 2 minutes, and equilibrate with filter paper in transfer buffer for 5 minutes; assemble the transfer clamps in sequence; transfer at 22 V for 30 minutes; wash the PVDF membrane in TBST solution for 10 minutes, and block with 5% skim milk powder at 37℃ for 2-3 hours; soak in TBST buffer 3 times, 15 minutes each time; add mouse-derived Anti-His primary antibody (10 μL / 50 mL deionized water) and incubate overnight at 4℃; soak in TBST buffer 4 times, 10 minutes each time; add goat anti-mouse anti-IgG antibody (5 μL / 50 mL deionized water) and shake at room temperature for 4-6 hours; soak in TBST buffer 4 times, 10 minutes each time; mix ECL luminescent developing solutions A and B 1:1 in the dark, drop evenly onto the PVDF membrane, and expose and develop using a protein luminescence analyzer.

[0055] The fusion protein "VapA-Linker-IHNV-G-6×His" with a molecular weight of 122 kDa was successfully constructed.

[0056] (5) Optimization of recombinant protein expression conditions: like Figures 5-7 As shown, the recombinant vector PGEX-4T-1-VapA-IHNV was transformed into E. coli competent cells BL21, and optimization experiments were conducted on temperature gradients (16℃, 20℃, 28℃, 32℃, 36℃), inducer concentration gradients (0, 0.2, 0.4, 0.6, 0.8, 1.0 mM), and induction time gradients (0, 6, 12, 18, 24, 30 h).

[0057] Take 1 mL of bacterial culture from each sample and sonicate to disrupt the protein. Separate the supernatant and precipitate. Analyze the expression of the recombinant protein under different conditions by SDS-PAGE electrophoresis.

[0058] The optimal induction conditions were determined to be: temperature 28℃, IPTG concentration 0.4 mM, and induction time 18 h.

[0059] Example 2: Fusion Protein Purification and Purification Yield Improvement (1) Purification of recombinant protein: The recombinant vector PGEX-4T-1-VapA-IHNV was induced to express 1 L of bacterial culture under optimal conditions. The bacterial cells were collected by centrifugation at 8000 rpm and 4℃ for 30 min, and then resuspended in 100 mL of PBS.

[0060] Homogenize for 5 cycles using a high-pressure homogenizer (power 40 Hz, pressure 1000 Pa), centrifuge at 8000 rpm and 4℃ for 30 min to separate the supernatant and precipitate. Remove impurities from the supernatant by passing it through a 0.45 μm filter membrane and store at 4℃.

[0061] Connect and fix the chromatography column to the UV detector, add 5 mL of Ni-ATK packing material, add 50 mL of Buffer I to equilibrate the nickel column and adjust the pH, repeat the washing process twice, and manually zero the column.

[0062] Add the protein solution to be purified, pass through the column twice, collect the flow-through, and control the flow rate to below 1 mL / min.

[0063] Add 50 mL of Buffer I to wash the column, repeat the washing process twice, until the reading remains unchanged.

[0064] Add Buffer II to elute impurities, adding 20 mL for the first time and 20 mL for the second time, and collect the eluent.

[0065] Add Buffer III to elute the target protein. For the first eluent, add 8 mL and slowly eluent, collect, and repeat twice. For the second eluent, add 4 mL and slowly eluent, collect, and repeat twice until the reading remains constant. Collect the 400 mM imidazole elution product.

[0066] The purity of the purified product was determined by SDS-PAGE electrophoresis.

[0067] (2) Fusion protein structure prediction and vector sequence optimization: Three-dimensional structural analysis of the fusion protein sequence using AlphaFold revealed highly overlapping regions at the fusion protein linking sites.

[0068] The full sequence of the vector was amplified using Escherichia coli (DH5α) with the recombinant vector PGEX-4T-1-VapA-IHNV as a template, with primers XB-for / XB-rev and the annealing temperature of the amplification system set to 90 s. The rest of the procedure was the same as described above.

[0069] The DNA single-stranded primers PGEX-3L-for / PGEX-3L-rev were diluted to 100 μmol / mL and mixed according to the following gradient annealing program: denaturation at 95°C for 2 min, annealing at 95°C for 8 s, with the annealing temperature decreasing by 0.1°C every 8 s for 700 cycles, and the reaction was terminated at 4°C for 7 min.

[0070] The recombinant vector sequence was seamlessly cloned and linked with the gradient annealing product. The reaction system consisted of 5 μL of One StepFusion Cioning Mix (2×), 4 μL of gradient annealing product, and 1 μL of recombinant vector sequence, for a total volume of 10 μL. The reaction conditions were 50℃ for 15 min.

[0071] The reaction product was transformed into E. coli DH5α, and positive transformations were selected for sequencing.

[0072] (3) Expression and purification after vector sequence modification: like Figure 8 As shown, the positively transformed cells with correct sequencing were subjected to plasmid extraction, transformation into BL21, and induced expression according to the above method.

[0073] Expression was induced for 18 h under optimal induction conditions, and the supernatant was collected after fragmentation and treated with Ni2+. + Purification by chromatography column.

[0074] The purified protein was identified and its concentration determined by Western blotting.

[0075] By extending the linker sequence by 3 times, the conformation of the fusion protein was optimized, the purification yield was improved, and a purified protein with a concentration of 0.822 mg / mL was finally obtained.

[0076] Example 3: Vaccine preparation and immunization efficacy evaluation (1) Vaccine preparation: The induction and expression of recombinant Escherichia coli were carried out under optimal conditions, and the inactivation method was based on Jin Huaiyuan (2021).

[0077] The purified fusion protein was mixed with adjuvant ISA763 at a volume ratio of 2:5 to achieve a final protein concentration of 100 μg / mL. The mixture was homogenized for 2 min using a high-speed homogenizer and then subjected to emulsification testing.

[0078] Inactivation test: Spread 200 μL of resuspended bacterial solution onto LB and TSA plates, and incubate at 37℃ and 20℃ respectively to observe whether there is colony growth.

[0079] The emulsification methods for inactivated recombinant Escherichia coli and inactivated C4 strain are the same as above.

[0080] (2) Fish immunity: Rainbow trout (weight 23.86±5.07 g) used in the experiment were randomly assigned to 4 groups, with 65 trout in each group: PBS group, bivalent subunit vaccine group, inactivated recombinant Escherichia coli group, and inactivated C4 group.

[0081] The experimental fish in each group were immersed in a solution of MS-222 anesthetic at a volume ratio of 1:200 with water, and aeration was carried out continuously during the anesthesia process.

[0082] The control group was injected with 0.1 mL PBS + adjuvant, while the immunized groups were injected with 0.1 mL emulsified protein, emulsified recombinant E. coli, and emulsified inactivated C4, respectively.

[0083] (3) Immunogenetic assay: Five fish were randomly selected from each group on days 3, 7, and 28 post-immunization. Kidney tissue was collected after anesthesia with MS-222 and stored at -80°C.

[0084] RNA was extracted from kidney tissue using an RNA extraction kit, and the RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0085] Using β-actin as an internal reference gene, qRT-PCR was performed to detect the immune genes (TLR3, stat, vig, mx, IL1β, IgM) in rainbow trout. The primers are shown in the table below. The reaction system consisted of: 10 μL SYBR™ Green Realtime PCR Master Mix, 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 7 μL ddH2O, and a total volume of 20 μL.

[0086] The relative quantitative data analysis employed the 2-ΔΔct method, and significance analysis and graphing were performed using GraphPad Prism 7.0.

[0087] like Figures 9-14 As shown in the figure, the results of kidney immunogene assays revealed that the IL-1β gene expression in the protein-immunized group and the C4-immunized group significantly increased on day 3 post-immunization (P < 0.05) and began to decline on day 7. However, the relative expression level of the recombinant E. coli-immunized group was significantly different from the control group on day 7 (P < 0.01), and the relative expression level was significantly higher in the recombinant E. coli-immunized group than in the control group on day 28 (P < 0.05). The TLR-3 gene expression in the kidneys of the protein-immunized group, the recombinant E. coli-immunized group, and the C4-immunized group all significantly increased on days 3, 7, and 28 post-immunization (P < 0.05). Overall, the relative expression level peaked on day 3 and declined on days 7 and 28. The levels of the kidney immunogene vig in the protein-immunized group were significantly different from those in the control group on days 3 and 7 post-immunization (P < 0.05), but no significant difference was observed on day 28. The levels in the recombinant E. coli-immunized group were significantly different from those in the control group on day 7 post-immunization (P < 0.05), but no significant difference was observed on days 3 and 28. No significant differences were observed in the C4-immunized group on days 3, 7, and 28. The levels of the kidney immunogene stat in the protein-immunized group were significantly different from those in the control group on day 3 post-immunization (P < 0.05), but no significant differences were observed on days 7 and 28. No significant differences were observed in the C4-immunized group on days 3, 7, and 28. The renal immunogene mx in the protein immunization group showed significant differences from the control group on days 3 and 7 post-immunization (P < 0.05), peaking on day 7, with no significant difference on day 28. The recombinant E. coli immunization group showed a significant difference only on day 7 (P < 0.05), with no significant differences on days 3 and 28. No significant differences were observed in the C4 immunization group. The renal immunogene igM in the protein immunization group, recombinant E. coli immunization group, and C4 immunization group showed significant differences from the control group on days 7 and 28 post-immunization (P < 0.05), with gene expression levels on day 28 all higher than on day 7, and no significant differences on day 3.

[0088] In summary, the expression levels of immune genes TLR3, stat, vig, mx, and IL-1β were significantly upregulated on day 3 post-immunization (P<0.05) and began to decline on day 7; IgM gene expression was significantly upregulated 28 days post-immunization (P<0.05).

[0089] (4) Neutralizing antibody titer determination: Cell resuscitation and passage: Remove the frozen EPC cells from the liquid nitrogen tank, thaw them rapidly in a 37°C water bath, wipe the outer wall of the cryovial with an alcohol swab, and place them in a biosafety cabinet. Slowly add the cell suspension to the culture flask and incubate in a 22°C CO2 incubator. Passage the cells every 3 days.

[0090] Virus titer assay: The IHNV virus stock solution was serially diluted 10-fold using cell maintenance medium to prepare 10... -1 Up to 10 -9 Nine dilutions of virus solution were prepared. EPC cells were digested and added to 96-well plates, cultured at 15°C until the cells adhered, and then different dilutions of virus solution were added. The cells were cultured at 15°C for 7 days, and cell cytopathic effects (CPE) were observed. The number of wells with cytopathic effects at each dilution was recorded, and the virus titer was calculated using the Reed-Muench method.

[0091] Neutralizing antibody titer assay: Twenty-eight days after immunization, ten rainbow trout were randomly selected from each group. Blood was collected from the tail vein, incubated at 4°C for 24 h, and then centrifuged to separate the serum. The serum was filtered through a 0.22 μm filter for sterilization. The serum was serially diluted twofold (1:4 to 1:256) with cell maintenance medium and mixed with an equal volume of 10-fold TCID50 IHNV virus solution. The mixture was incubated at 37°C for 1 hour. The virus-serum mixture was inoculated into 24-well EPC cell plates, 1 mL per well, with four replicates per dilution. The plates were incubated at 15°C for 7 days. The number of wells showing typical CPE was recorded, and the serum neutralizing antibody titer was calculated using the Reed-Muench method. The results are shown in the table below. like Figure 15 As shown, specific neutralizing antibodies were detected in the serum 28 days after immunization.

[0092] (5) Infection challenge experiment: Twenty-eight days after immunization, 30 rainbow trout from each group were infected with Aeromonas salmonicidae bacterium solution by immersion at a concentration of 1×10⁻⁶. 7 CFU / mL.

[0093] The mortality rate was statistically observed over 14 days, and the relative immunization rate was calculated using the formula RPS=[1-(vaccinated mortality rate / unvaccinated mortality rate)]×100%.

[0094] The results are shown in the table below: In summary, the vaccine of this invention provides 100% immune protection against Aeromonas salmonicida.

[0095] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A bivalent subunit vaccine against Aeromonas salmonicida and infectious hematopoietic necrosis virus, characterized in that, It contains a fusion protein of Aeromonas salmonidae antigen and infectious hematopoietic necrosis virus antigen, as well as an immune adjuvant.

2. The bivalent subunit vaccine according to claim 1, characterized in that, The structure of the fusion protein is VapA-Linker-IHNV-G-6×His, and the specific sequence is shown in SeqAA VapA-IHNV.

3. The bivalent subunit vaccine according to claim 1, characterized in that, The Aeromonas salmonidae antigen and the infectious hematopoietic necrosis virus antigen are fused together via a flexible linker.

4. The bivalent subunit vaccine according to claim 3, characterized in that, The flexible linker is GGGGS.

5. The bivalent subunit vaccine according to claim 1, characterized in that, The vaccine contains 50-150 μg / mL of fusion protein and adjuvant ISA763.

6. The method for preparing the bivalent subunit vaccine according to any one of claims 1-5, characterized in that, The process includes the following steps: constructing a fusion protein expression vector, transforming the expression host, inducing the expression of the fusion protein, purifying the fusion protein, and emulsifying the purified fusion protein with an adjuvant.

7. The preparation method according to claim 6, characterized in that, The conditions for inducing expression were: temperature 28℃, IPTG concentration 0.4mM, and induction time 18h.

8. The preparation method according to claim 6, characterized in that, The purified fusion protein includes the use of Ni 2+ Purification was performed using an affinity chromatography column.

9. The preparation method according to claim 6, characterized in that, The construction of the fusion protein expression vector involves inserting the VapA gene and the IHNV-G gene into the expression vector PGEX-4T-1 via homologous recombination technology.

10. The use of the bivalent subunit vaccine as described in claim 1 in the prevention of Aeromonas salmoneri and infectious hematopoietic necrosis virus infection.

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